Williams, Harry Michael ORCID: https://orcid.org/0000-0001-9108-9933 (2021) Structural characterisation of the human innate immune proteins FIBCD1 and surfactant proteins A and D. Doctoral thesis, Keele University.

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Abstract

The surfactant proteins A and D (SP-A and SP-D) and fibrinogen C containing domain 1 (FIBCD1) are pattern recognition receptors, which recognise pathogens through the binding of carbohydrate residues embedded within surface structures. The work presented here characterises, through X-ray crystallography, the recognition of carbohydrate structures using recombinant fragments of human SP-D (rfhSP D) and FIBCD1. Additionally, a fragment of human SP-A has been produced for crystallisation studies.
The crystal structures of FIBCD1 in complex with N acetylalanine, N-acetylglucosamine (GlcNAc), N acetylgalactosamine-4-sulfate (GalNAc-4S), N acetylneuraminic acid (Neu5Ac), and (GlcNAc)2 have been solved by molecular replacement. A new native structure has also been solved by molecular replacement. The structures are refined to 1.84 – 2.00 Å. These structures reinforce our current understanding of ligand recognition by FIBCD1 but also highlight an extended ~25 Å binding surface upon which extended oligosaccharides may bind.
The crystal structures of rfhSP-D and four synthetic oligosaccharides have also been solved by molecular replacement. The oligosaccharides, designated HepI-HepII HepIII, HepI-KdoI, HepIPhos- HepII, and Phos-HepII-HepI, correspond to the core oligosaccharides of Haemophilus influenzae and Salmonella enterica LPS. The structures are refined to 1.63 – 1.92 Å. The ligand-bound rfhSP-D structures clarify SP-D recognition of the core HepI-KdoI motif and highlight an elegant mechanism by which bacteria may use phosphorylation of core oligosaccharide residues to evade immune detection.
A human SP-A neck/carbohydrate recognition domain (N/CRD) has also been produced using a type IV collagenase enzyme isolated from Clostridium histolyticum. The cleaved SP-A N/CRD fragment has a monomeric molecular mass of ~18 kDa and elutes as a trimer from a size-exclusion chromatography column. This fragment could be used in future structural studies so as to better understand how human SP-A operates. Ultimately, this work has led to a better understanding as to how FIBCD1, SP-A, and SP-D operate and function as part of the innate immune response.

Item Type: Thesis (Doctoral)
Subjects: Q Science > QH Natural history > QH301 Biology
Divisions: Faculty of Natural Sciences > School of Life Sciences
Contributors: Shrive, AK (Thesis advisor)
Greenhough, TJ (Thesis advisor)
Depositing User: Lisa Bailey
Date Deposited: 17 Mar 2021 12:31
Last Modified: 17 Mar 2021 12:35
URI: https://eprints.keele.ac.uk/id/eprint/9262

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